The long-range goal of our studies is to understand the fundamental mechanisms of protein-DNA recognition by correlating high-resolution structures of protein/DNA complexes with functional studies. In this proposal, we will address how conserved core DNA binding elements are modulated to recognize divergent DNA sequences. To do this, we have chosen three different model systems to address three distinct aspects of this type of modulation of DNA-binding that underlies many protein-DNA recognition systems. In Aim 1 we will address how a family of fungal specific proteins use different dimerization domains and "linker" regions with a conserved Zn2Cys6 binuclear cluster domain to recognize conserved CGG half sites that differ in the polarity and inter-base spacing of these half sites. We have previously determined the structures of DNA complexes with the Gal4, Ppr1, Put3, Hap1 and Leu3 members of this family. We will now extend our studies to the Pdr1 and PrnA members and continue our studies with the Gal4 and Leu3 member of the family to address additional aspects of DNA site selection by these proteins including a structure-based mutational analysis to interchange their DNA-binding specificities. For Aim 2 we will use the p53 tumor suppressor and DNA-binding protein as a model to understand how protein multerimerization mediates cooperative DNA-binding. In vivo, p53 binds DNA as a cooperative dimer of dinners. We have recently employed a crosslinking strategy to "trap" a crystal structure of a p53 dimer bound to DNA and we will now use a similar strategy to prepare a dimer of p53 dimers bound to DNA. For Aim 3 we will use the FOXO longevity protein and DNA binding protein to probe how posttranslational acetyl- and phospho- modifications within a DNA-binding domain modulate its DNA-binding properties. Together, the studies proposed here will provide new insights into the fundamental mechanisms underlying the modulation of protein/DNA recognition. The importance of these studies are underscored by the fact that all DNA transactions such as transcription, replication and DNA repair depend on the faithful protein-mediated recognition of DNA and that the perturbation of this process, as occurs with p53 and FOXO, is often correlated with human diseases such as cancer. These studies may therefore provide therapeutic avenues for the treatment of disorders mediated by defects in aspects of protein-DNA recognition.